The goal of this project is to develop a gene therapeutic for retinitis pigmentosa (RP). RP is the most common inherited form of blindness, and is characterized by photoreceptor loss, leading to a progressive loss of peripheral as well as night vision. There is no treatment for RP, which is a group of diseases caused by mutations in one of multiple genes that lead to similar phenotypes. X-linked retinitis pigmentosa (XLRP) is a form of RP that is most often caused by mutations in a gene encoding the photoreceptor protein RPGR (retinitis pigmentosa GTPase regulator), and is relatively common, accounting for approximately 10% of all cases of RP. Most mutations in RPGR are recessive, indicating that gene replacement can be an effective strategy. Recombinant adeno-associated virus (AAV) vectors have been showing great promise in delivering genes in animal models of retinal disease, giving long-term, stable gene expression without inflammation. Novel AAV serotypes including AAV8 have been identified which target the retinal pigment epithelium (RPE) or the photoreceptor layer, and transduce those cell types with higher efficiency than previously known serotypes of AAV. This project proposes to use AAV8, which excels at delivering genes to photoreceptors, to develop a therapeutic for XLRP. Initially, optimization studies will be performed to identify the most effective clinical candidate for RPGR gene replacement, and then safety and toxicology studies will be performed with the proposed clinical candidate, with the goal of filing an IND by the end of the project period. Initially, several alternative isoforms of the RPGR cDNA will be tested in the mouse model of XLRP, the RPGR knockout mouse, to determine which is the most effective in preventing retinal degeneration. When the optimal cDNA is selected, the promoter will then be optimized, testing both a constitutive (CMV) promoter and a photoreceptor- specific (rhodopsin) promoter to determine if one promoter has an efficacy advantage over the other. The promoters will also be used with a reporter gene in normal mice and non-human primates to confirm that both are stable for long-term transgene expression. The final vector construct will also be used to determine the minimal dose of vector required for biologic effect in the RPGR knockout mouse. Finally, the clinical candidate, with the AAV8 serotype, and optimized promoter and RPGR transgene will be put through biodistribution and safety/toxicology studies in mice and in dogs to complete the package of data needed for filing an IND. The proposed gene therapeutic for XLRP will serve an unmet medical need in RP patients, and will be a novel "first-in-class" therapy that will provide a platform for treating additional forms of RP and other retinal diseases. PUBLIC HEALTH RELEVANCE: Retinitis pigmentosa is the most common inherited form of blindness, with an estimated 100,000 patients in the US. It is characterized by progressive loss of peripheral as well as night vision, eventually resulting in blindness. This proposal is designed to develop a gene therapeutic that will provide significant medical relief to patients, develop technologies applicable to other indications in the eye, and minimize the burden on the healthcare system.